ATP-dependent roteases are a unique family of proteolytic enzymes that require energy to unfold protein substrates prior to peptide bond hydrolysis. In the cytosol and nucleus, the 26S proteasome is the only ATP-dependent protease, which functions to eliminate abnormal and damaged proteins, and to regulate cell differentiation, cell cycle progression, and the immune response. The proteasome has emerged as an important drug target in the treatment of multiple myeloma, and possibly other cancers. Within mitochondria, there are two soluble ATP- dependent proteases in the matrix-ClpXP and Lon. Our unpublished work suggests that these mitochondrial proteases are novel targets for drug discovery, and that inhibitors of these enzymes, either alone or in combination with proteasome inhibitors, may provide new and targeted therapeutic strategies for the treatment of cancer. In addition, activators of mitochondrial ATP-dependent proteolysis may have therapeutic potential in the treatment of neurodegenerative and/or myocardial dysfunctions that are linked to mitochondrial protein aggregation. The goal of this project is to develop high throughput screening (HTS) assays to identify small molecule inhibitors and activators of ClpXP, and to distinguish small molecules that target ClpXP versus Lon. These two ATP-dependent proteases have fundamentally distinct roles in the cellular response to stress and environmental adaptation, and thus represent distinct drug targets. For example, ClpXP expression is induced by the accumulation of unfolded proteins within mitochondria, whereas Lon expression is unchanged. However, Lon but not ClpXP expression is upregulated by unfolded proteins within the endoplasmic reticulum (ER), and is likely important in the ER stress response, which is important in numerous diseases states and aging. As ClpXP and Lon have fundamentally different cellular functions, it is essential to identify compounds that specifically target these enzymes, if we are to understand and exploit their therapeutic potential. ClpXP and Lon proteases are soluble and readily purified with high yield, making them suitable for high throughput biochemical assays. We have already made substantial progress in developing high throughput screening assays for the Lon protease. Thus, this project aims at developing a rigorous set of high throughput screening assays optimized for the ClpXP protease. (1) We will develop and optimize a primary screen that specifically measures the protease activity of purified ClpXP. We will also optimize secondary- and counter-screening assays for characterizing and prioritizing active compounds that target ClpXP. Secondary screens will determine the effect of active compounds on the ATPase and peptidase activity of ClpXP;and counter screens will determine the specificity of active compounds by measuring their effects on Lon and/or the proteasome. (2) In addition, we will develop a high-content screening assay for ClpXP-dependent proteolysis using a ClpXP reporter substrate. Using this cell-based assay we will determine whether compounds identified in the primary screen are membrane-permeable, and whether under physiological conditions they activate or inhibit ClpXP-mediated degradation of a mitochondrial reporter substrate. Taken together, the primary, secondary, and counter screens, in combination with a cell-based assay will permit us to identify, prioritize and validate active compounds that target the mitochondrial ClpXP protease. PUBLIC HEALTH RELEVANCE: The aim of this one-year R21 project is to develop a high-throughput screening (HTS) program for identifying small molecules, which specifically and potently inhibit or activate the ClpXP ATP- dependent protease. A rigorous set of biochemical and cell-based assays that are amenable to HTS, will measure the protease and ATPase activities of ClpXP. These assays will be used to discriminate small molecules that target the ClpXP protease from the Lon protease, which is the only other ATP-dependent protease within the mitochondrial matrix. Identifying small molecule inhibitors or activators will be the first step in developing compounds that permit us to discrimate the physiological functions of ClpXP and Lon, and will assist in defining ClpXP and Lon as potential clinical targets in mitochondrial dysfunction, which is associated with numerous neuromuscular disorders, cardiomyopathies, cancers and aging. Inhibitors of ClpXP and/or Lon have likely application as anti-cancer agents used either alone or in combination with other chemotherapeutic strategies. This possibility is supported by preliminary data demonstrating that ClpXP and Lon are up-regulated in a variety of lymphoma cell lines, and that compounds used to treat leukemia and solid tumors also inhibit Lon-mediated proteolysis. In addition, activators of ClpXP and/or Lon may also be exploited in the treatment of neurodegenerative disorders linked to mitochondrial protein aggregation.